Membrane receptor proteins located in epithelial cells of insect midguts are target sites for the action of crystalline protein toxins (cry toxins) produced by Bacillus thuringiensis (Bt). Activated cry toxins bind to insect receptor proteins on insect midgut epithelial cells and cause toxicity and death to specific insect pests. The specific interactions between the receptor and toxin are key events in discerning the structure-activity relationships and modes of action of these cry toxins. Being able to measure such interactions is greatly facilitated by the recombinant over production and purification of these receptor proteins to produce sufficient quantities and to reduce the level of non-specific interactions from host impurities. Using purified, functional receptor proteins in biochemical studies allows the measurement of binding affinities and specificities of the receptors in interactions with a range of Bt toxins. However, the membrane receptor proteins are typically hydrophobic, and thus are not soluble in common buffers that are suitable for most protein extractions. A specific purification protocol is described here that maximizes the solubility of hydrophobic insect membrane-bound proteins while preserving their function as receptors to toxins.
There are many reports of purifying membrane-bound proteins from recombinant hosts using N-laurylsarcosine (N-Dodecyl-N-methylglycine). See, for example, references [1-4]. However, none describes the use of a 1.0% to 0.1% gradient which the inventors found to be surprisingly effective at maximizing the solubility and activity of some hydrophobic membrane-bound receptors found in insect intestinal cell that are produced in recombinant Spodoptera frugiperda (Fall Armyworm) Sf9 insect cells. This method maximally solubilizes membrane receptor proteins for high yield, promotes column binding, and blocks protease cleavage. The purified receptors through this method have high purity and have retained biochemical and biological activity.